The Blues, trailing to a goal from Valencia winger Rodrigo, were given a lifeline in the closing stages when VAR flagged up a handball in the area.
But Barkley’s spot-kick clipped the crossbar to condemn Lampard and his young side to a 1-0 loss in the Group H opener.
The Spaniards were supposed to be there for the taking, having arrived in west London in turmoil following the sacking of popular boss Marcelino last week, a move which has angered the players as well as the fans.
But they did not play like a team on the verge of mutiny and had too much nous for Lampard’s beginners, four of whom were making their Champions League debuts.
Lampard, who played 102 Champions League matches for Chelsea and captained them to their 2012 triumph, was perfectly placed to impart his experience of playing at this level to his youngsters – but it could still be a steep learning curve this season.